ES2959412T3 - Stabilized tin compositions - Google Patents

Abstract

The present invention relates to improved amorphous calcium phosphate and/or amorphous calcium fluoride phosphate complexes stabilized by phosphopeptides/phosphoproteins by the addition of stannous ions. These complexes have anticariogenic properties useful in protecting dental structures, as they remineralize (repair) the early stages of dental caries and have other dental/medical applications (including anticalculus, anti-erosion/corrosion, and antidentin hypersensitivity). Also provided are methods for preparing the complexes of the invention and for the treatment or prevention of various dental conditions including dental caries, dental calculus, dental erosion/corrosion and dental hypersensitivity. (Automatic translation with Google Translate, without legal value)

Description

DESCRIPTION

Stabilized tin compositions

field of invention

The present invention relates to improved amorphous calcium phosphate and/or amorphous calcium fluoride phosphate complexes stabilized by phosphopeptides/phosphoproteins by the addition of stannous ions. These complexes have anticariogenic properties useful in protecting dental structures, as they remineralize (repair) the early stages of dental caries and have other dental/medical applications (including anticalculus, anti-erosion/corrosion, and antidentin hypersensitivity). Also provided are methods for preparing the complexes of the invention and for the treatment or prevention of various dental conditions including dental caries, dental calculus, dental erosion/corrosion and dental hypersensitivity.

This application claims priority to Australian patent applications no. 2013905081, 2014901202 and 2014903815.

Background of the invention

Common causes of hypomineralized lesions are caries and fluorosis.

Dental caries is initiated by the demineralization of the hard tissue of the teeth, generally by organic acids produced from the fermentation of dietary sugar by odontopathogenic bacteria in dental plaque. Dental caries remains a major public health problem. Additionally, restored tooth surfaces may be susceptible to further dental caries around the margins of the restoration. Although the prevalence of dental caries has decreased thanks to the use of fluoride in most developed countries, the disease remains a major public health problem. Dental erosion or corrosion is the loss of dental minerals due to dietary or regurgitated acids. Dental hypersensitivity is due to the exposure of the dentinal tubules through the loss of the protective mineralized layer, cementum. Dental calculus is the unwanted buildup of calcium phosphate minerals on the surface of the tooth. All of these conditions, dental caries, dental erosion, dental hypersensitivity and dental calculus are therefore imbalances in the level of calcium phosphates.

Enamel fluorosis (mottle) has been recognized for almost a century; however, the etiological role of fluoride was not identified until 1942 (Black and McKay, 1916). The characteristic appearance of fluorosis can be differentiated from other enamel disorders (Fejerskovet al., 1991). The clinical characteristics of fluorotic enamel lesions (FLE) represent a continuum ranging from fine opaque lines following the perikymata to calcareous white enamel (Fejerskovet al.,1990; Giambroet al.,1995). The presence of a comparatively highly mineralized outer enamel surface and a hypomineralized subsurface in the fluorotic lesion simulates the incipient carious lesion of the enamel "white spot" (Fejerskovet al., 1990). As severity increases, both the depth of enamel affected by the lesion and the degree of hypomineralization increase (Fejerskovet al.,1990, Giambroet al.,1995). The development of fluorosis is highly dependent on the dose, duration and timing of fluoride exposure (Fejerskovet al.,1990, Fejerskovet al.,1996; Aoba and Fejerskov, 2002) and is believed to be related to elevated concentrations. of fluoride in serum. Chalky "white spot" lesions may also form on children's developing teeth, such as after treatment with antibiotics or fever. These lesions indicate areas of hypomineralization of the tooth enamel.

Depending on the severity of the lesion, fluorosis has been managed clinically by restorative replacement or microabrasion of the external enamel (Den Besten and Thariani, 1992; Fejerskovet al., 1996). These treatments are unsatisfactory because they involve restorations or removal of dental tissue. What is desired is a treatment that mineralizes hypomineralized enamel to produce a natural appearance and structure.

Specific complexes of casein phosphopeptides and amorphous calcium phosphate ("CPP-ACP", commercially available as Recaldent™) have been shown to remineralize subsurface enamel lesions in vitro and in situ (Reynolds, 1998; Shenet al., 2001; Reynolds et al. ,2003).

WO 98/40406 in the name of The University of Melbourne describes casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) complexes and CPP-stabilized amorphous calcium fluoride phosphate complexes (CPP-ACFP) that have been produced at alkaline pH. Such complexes have been shown to prevent enamel demineralization and promote remineralization of subsurface enamel lesions in animal and human caries models (Reynolds, 1998). CPPs that are active in complex formation do so regardless of whether or not they are part of a full-length casein protein. Examples of active (CPP) that can be isolated after tryptic digestion of full-length casein have been specified in US Patent No. 5,015,628 and include Bos as<1>-casein X-5P peptides ( f59-79) [1], Bos p-casein X-4P (f1 -25) [2], Bos as<2>-casein X-4P (f46-70) [3] and Bos as<2>-casein X-4P (f1 -21) [4]. Furthermore, improvements in these compositions are disclosed in WO 2006/056013 and Wo 2007/090242 and specific uses.

EP 1525878 describes a caries prevention composition comprising a casein phosphopeptide-amorphous calcium phosphate complex (CPP-ACP) and/or a casein phosphopeptide-amorphous calcium fluoride phosphate complex (CPP-ACFP), a sodium carboxymethylcellulose having a degree of etherification, a viscosity regulator, and a residual amount of water.

Stannous fluoride (SnF<2>) is one of several metal fluoride salts that have been proposed as an anti-caries and anti-plaque agent; However, there are difficulties in administering this agent in toothpastes, mouthwashes, and other oral care products due to the instability of stannous ions. In the presence of hydroxide ions and phosphate ions, tin can precipitate as stannous hydroxide and stannous phosphate complexes. This precipitate is significantly less biologically available and can be more easily removed from the oral cavity by swallowing. Furthermore, stannous ions can be oxidized to stannic ions (SnIV), which are even more reactive to produce poorly soluble forms with very low activity. New SnF<2>toothpaste formulations rely on being non-aqueous or using polyphosphate to stabilize stannous ions. However, these formulations have little acceptance, especially among people with dry mouth. There is a need to provide improved or alternative treatments for hypomineralized lesions.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of common general knowledge in Australia or any other jurisdiction or that it could be expected to be prior art is verified, understood or considered relevant by a person skilled in the art.

Summary of the invention

The invention refers to the subject of the claims.

In one aspect, the invention provides an amorphous calcium phosphate (ACP) complex stabilized with tin-associated phosphopeptide and/or amorphous calcium fluoride phosphate (ACFP) complex having a stannous ion content of at least 1 mol of tin per mole of phosphopeptide. Tin can bind to phosphopeptide-stabilized amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) as determined using the experimental protocol in Example 2. In one embodiment, the phosphopeptide-stabilized amorphous calcium phosphate Tin-associated phosphopeptide (ACP) and/or amorphous calcium fluoride phosphate complex (ACFP) are produced by the method described herein, including, but not limited to, the method described in Example 1.

Without being bound by any theory or mode of action, it is believed that phosphopeptides, such as casein phosphopeptides, can stabilize a stannous compound in an aqueous environment and in the presence of stabilized amorphous calcium phosphate (ACP) and calcium fluoride phosphate. Stabilized amorphous calcium (ACFP) These complexes are superior to other forms of fluoride and stabilized ACP or ACFP in remineralizing subsurface enamel lesions. The mineralization of tooth surfaces can be significantly improved by providing a stannous compound during the mineralization process using stabilized ACP and/or stabilized ACFP. In particular, enamel mineralization by stabilized soluble forms of tin-associated ACP complexes and tin-associated ACFP complexes has been found to be increased compared to stabilized ACP and fluoride without associated tin. In other words, stannous ions form complexes with CPP-ACP and/or CPP-ACFP complexes, and these Sn-associated CPP-AC(F)P complexes offer superior properties. Various compositions incorporating these complexes are useful for administration. When the stannous salt of fluoride is used, additional fluoride ions are available in compositions of stannous-associated ACP/ACFP complexes. Additional fluoride ions can also be provided by including NaF in the composition.

The invention provides a tin-associated phosphopeptide-stabilized ACP or ACFP having a stannous ion content of at least 1 mole of tin per mole of phosphopeptide. Preferably, the tin-associated phosphopeptide-stabilized ACP or ACFP has a stannous ion content of at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 moles of tin per mole of phosphopeptide. Even more preferably, the stannous ion content is in the range of 1 to 100, 1 to 50, 1 to 20 or 1 to 10 moles of tin per mole of phosphopeptide. The present invention also provides a composition comprising, or consisting of, ACP and/or ACFP complexes stabilized with stannous-associated phosphopeptides as described herein.

In any embodiment, the above stannous ion content may be the content of stannous ions closely bound to the complex (as described herein). In evaluating the content of stannous ions, the content of closely bound stannous ions is measured by the methods described herein, in particular, in Example 6.

The invention also provides a tin-associated phosphopeptide-stabilized ACP and/or ACFP complex comprising stannous ions that remain associated with the complex after centrifugation on a molecular weight cutoff filter of 1,000 to about 3,000 g for 1 hour at room temperature.

The invention also provides a tin-associated phosphopeptide stabilized ACP or ACFP having at least 50, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 , 99 or 100% of the tin associated with the complex as tightly bound as determined by the method of Example 6.

The invention also provides a tin-associated phosphopeptide-stabilized ACP or ACFP having at least , 99 or 100% of the tin used in the preparation of the complexes incorporated into the complexes. The complex can be prepared as described in Example 1.

In one embodiment, the invention provides an amorphous calcium phosphate (ACP) stabilized with phosphopeptide associated with stannous fluoride and/or an amorphous calcium fluoride phosphate (ACFP) complex associated with stannous fluoride. In another aspect, the invention provides an oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP and/or ACFP. In any embodiment, the stannous compound is preferably stannous fluoride and/or stannous chloride. NaF is also optionally included.

In one aspect, the present invention provides a tin-associated phosphopeptide-stabilized ACP or ACFP for use in a method of mineralization of a tooth surface or subsurface comprising contacting the tooth surface or subsurface with (a) a formulation for the oral care that includes an ACP and/or ACFP stabilized with tin-associated phosphopeptide, or (b) an ACP or ACFP stabilized with tin-associated phosphopeptide.

The tin-containing compound may be any soluble tin-containing compound suitable for oral use. Preferably, the tin-containing compound is a stannous salt. Tinnous salt may contain fluoride. A stannous salt includes, but is not limited to, stannous fluoride, stannous chloride, potassium stannous fluoride, sodium stannous fluorozirconate, stannous chloride fluoride, stannous acetate, sodium stannous fluoride, stannous hexafluorozirconate, stannous sulfate, stannous tartrate, stannous gluconate, citrate disodium monotin. Preferred stannous salts include stannous fluoride and stannous chloride.

The tooth surface is preferably tooth enamel or dentin. In one embodiment, the tooth surface is a lesion in the enamel, such as a lesion caused by caries, dental erosion or fluorosis. In another embodiment, the surface is exposed dentin causing dentin hypersensitivity.

Stabilized amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) are stabilized with phosphopeptide. Preferably, the phosphopeptide (as defined below) is a casein phosphopeptide.

In a preferred embodiment, the phosphopeptide-stabilized amorphous calcium phosphate (ACP) complex or amorphous calcium fluoride phosphate (ACFP) has strongly bound and weakly bound calcium, wherein the bound calcium in the complex is less than the strongly bound calcium. bound into an ACP or ACFP complex formed at a pH of 7.0. Optionally, the ACP or ACFP is predominantly presented in a basic form.

In another aspect, the invention also provides a composition including ACP or ACFP stabilized with stannous phosphopeptide, further comprising fluoride, wherein the fluoride is provided as stannous fluoride and sodium fluoride. Preferably, the composition includes 2% w/v stabilized ACP or ACFP, about 1,100 ppm fluoride as stannous fluoride and about 350 ppm fluoride as sodium fluoride, or the composition includes 0.4% w/v ACP or Stabilized ACFP, approximately 220 ppm fluoride as stannous fluoride and approximately 70 ppm fluoride as sodium fluoride. Preferably, the composition is a toothpaste.

In a preferred embodiment, the calcium ion content of the phosphopeptide-stabilized ACP or ACFP complex is in the range of about 30 to 100 moles of calcium per mole of PP. More preferably, the calcium ion content is in the range of about 30 to about 50 moles of calcium per mole of PP.

In a preferred embodiment, the ACP and/or ACFP is in the form of a complex of ACP and/or ACFP stabilized with casein phosphopeptide.

Preferably, the ACP phase is primarily (i.e., >50%) a basic phase, wherein the ACP predominantly comprises the Ca2+, PO<4>3- and OH- species. The basic phase of ACP may have the general formula [Ca3(PO4)2]X[Ca2(PO4)(OH)] where x > 1. Preferably x = 1-5. More preferably, x = 1, that is, the two components of the formula are present in equal proportions. Accordingly, in one embodiment, the ACP base phase has the formula Ca3(PO4)2Ca2(PO4)(OH).

Preferably, the phase of the ACFP is primarily (i.e., >50%) basic phase, wherein the ACFP predominantly comprises the Ca2+, PO<4>3- and F- species. The basic ACFP phase may have the general formula [Ca3(PO4)2]X[Ca2(PO4)F]y where x > 1 when y = 1 or where y > 1 when x = 1. Preferably, y = 1 and x = 1-3. More preferably, y = 1 and x = 1, that is, the two components of the formula are present in equal proportions. Accordingly, in one embodiment, the base phase of ACFP has the formula Ca3(PO4)2Ca2(PO4)F.

In one embodiment, the ACP complex consists essentially of phosphopeptides, calcium, phosphate and hydroxide ions and water.

In one embodiment, the ACFP complex consists essentially of phosphopeptides, calcium, phosphate, fluoride and hydroxide ions and water.

In a further aspect of the present invention, there is provided a tin-associated phosphopeptide-stabilized ACP or ACFP for use in a method of mineralization of a tooth surface comprising providing (a) an oral care formulation that includes an ACP and /or ACFP stabilized with tin-associated phosphopeptide, or (b) an ACP or ACFP stabilized with tin-associated phosphopeptide. In a preferred embodiment, the tooth surface is enamel.

In a further aspect of the present invention, there is provided a tin-associated phosphopeptide-stabilized ACP or ACFP for use in a method of forming a coating on the surface of a tooth comprising providing (a) an oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP and/or ACFP, or (b) a tin-associated stabilized ACP or ACFP. Preferably, the tooth is located in a subject identified as susceptible to, or suffering from, any one or more of dental erosion, demineralization, caries or dentin hypersensitivity. Stabilized amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) are stabilized with phosphopeptide. Preferably, the phosphopeptide (as defined below) is a casein phosphopeptide.

Said layer can be characterized as having a calcium:phosphate ratio equivalent to normal apatite, preferably when the ratio is approximately 2:1. Ideally, the layer contains an amount of calcium of approximately 20% by weight.

Preferably, the layer has approximately 3 to 12 times more stannous ions than healthy enamel.

Preferably, the layer contains carbon, oxygen, fluoride, phosphate, calcium and tin. The layer may have approximately an elemental analysis of 20-30% by weight of carbon, 35-45% by weight of oxygen, 0.1-1% by weight of fluoride, 8 to 12% by weight of phosphate, 16 to 24 % by weight of calcium and/or 0.5-2% by weight of tin. Alternatively, the layer may have an elemental analysis of any one of the elements, such as calcium, phosphate, fluoride, carbon and/or tin, as shown in any one of Tables 3 or 4.

In a further aspect of the present invention, a tin-associated phosphopeptide-stabilized ACP or ACFP is provided for use in a layer-forming process having a calcium:phosphate ratio equal to, or close to, that of apatite. normal on a tooth surface, the process comprising contacting the tooth surface with (a) an oral care formulation that includes an ACP and/or ACFP stabilized with tin-associated phosphopeptide, or (b) an ACP or ACFP stabilized with tin-associated phosphopeptide.

Provided is (a) An oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP and/or ACFP for use in a method of protecting a tooth surface, or (b) an associated phosphopeptide-stabilized ACP or ACFP to tin for use in a method of protecting a tooth surface. Typically, the tooth surface may be one that has been identified as beneficial from a superficial layer, for example, due to a greater likelihood of demineralization. The tooth surface can be an enamel surface or a dentin surface.

In a further aspect of the present invention, a tin-associated phosphopeptide-stabilized ACP or ACFP is provided for use in a method of treating fluorosis comprising contacting a fluorotic lesion in dental enamel with (a) a formulation for oral care that includes an ACP and/or ACFP stabilized with tin-associated phosphopeptide, or (b) an ACP or ACFP stabilized with tin-associated phosphopeptide.

In a further aspect of the present invention there is provided (a) an oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP and/or ACFP, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP for its use in a method for treating dental caries comprising contacting a carious lesion in dental enamel with a tin-associated phosphopeptide-stabilized ACP or ACFP.

In a further aspect of the present invention, a tin-associated phosphopeptide-stabilized ACP or ACFP is provided for use in a method of treating dental erosion comprising contacting a lesion in the dental enamel caused by erosion with (a ) an oral care formulation that includes an ACP and/or ACFP stabilized with tin-associated phosphopeptide, or (b) an ACP or ACFP stabilized with tin-associated phosphopeptide.

In a further aspect of the present invention, a tin-associated phosphopeptide-stabilized ACP or ACFP is provided for use in a method of reducing dentin hypersensitivity comprising contacting exposed dentin with (a) an oral care formulation. including a tin-associated phosphopeptide-stabilized ACP and/or ACFP, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP.

In a further aspect of the present invention, a tin-associated phosphopeptide-stabilized ACP or ACFP is provided for use in a method of remineralizing a lesion in dental enamel or dentin comprising contacting the lesion with (a) a oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP and/or ACFP, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP The tin-associated phosphopeptide-stabilized ACP or ACFP is stabilized by a phosphopeptide ( PP). In a preferred embodiment, the phosphopeptide is a casein phosphopeptide. Preferably, the ACP or ACFP is in the form of a complex of ACP or ACFP stabilized with casein phosphopeptide.

In any aspect or embodiment as described herein, the tin-associated phosphopeptide-stabilized ACP or ACFP may be in a formulation with additional calcium phosphate. Typically, the formulation includes a PP-stabilized ACP and/or ACFP complex together with at least an equal amount by weight of calcium phosphate.

In a further aspect of the invention, a tin-associated phosphopeptide-stabilized ACP or ACFP is provided for use in a method of remineralizing a lesion in dental enamel comprising contacting the lesion with (a) a care formulation buccal including a tin-associated phosphopeptide-stabilized ACP and/or ACFP, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP followed by administration of a composition containing sodium bicarbonate or urea. Preferably, the composition is a mouthwash containing sodium bicarbonate or urea.

In any aspect or embodiment of the invention described herein, a compound that is capable of increasing or maintaining the pH of a solution can be administered simultaneously with, as a pretreatment of, or as a posttreatment of (a) a skin care formulation. buccal that includes a stabilized ACP and/or ACFP associated with tin, or (b) an ACP or ACFP stabilized with phosphopeptide associated with tin.

In any aspect or embodiment of the invention described herein, (a) an oral care formulation that includes a tin-associated stabilized ACP and/or ACFP, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP , can be applied to the mouth, tooth or injury by the subject needing treatment or by a dental health professional.

An oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP or ACFP can be contacted with the tooth surface for a period of about 1 to 60 minutes, or for about 1 to 30 minutes. In one embodiment, a composition including a stannous compound and stabilized ACP/ACFP is contacted with the tooth surface for approximately 20 minutes.

Preferably, an oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP or ACFP is contacted with the tooth surface for a period of about 1 minute to 2 hours, or 5 minutes to 60 minutes, or about 10 minutes. . The oral care formulation including a tin-associated phosphopeptide-stabilized ACP/ACFP can be repeatedly applied to the tooth surface over a period of 1 day to several months.

A tin-associated phosphopeptide-stabilized ACP or ACFP can be contacted with the tooth surface for a period of about 1 to 60 minutes, or for about 1 to 30 minutes. In one embodiment, a tin-associated phosphopeptide-stabilized ACP/ACFP is contacted with the tooth surface for approximately 20 minutes.

Preferably, a tin-associated phosphopeptide-stabilized ACP or ACFP is contacted with the tooth surface for a period of about 1 minute to 2 hours, or 5 minutes to 60 minutes, or about 10 minutes. Tin-associated phosphopeptide-stabilized ACP or ACFP can be applied repeatedly to the tooth surface over a period of 1 day to several months.

In one embodiment, (a) an oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP and/or ACFP, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP, is contacted with the surface. dental from 1 to 60 minutes, or from 1 to 30 minutes or from 1 to 5 minutes.

In one embodiment, the tooth surface needs such treatment. Therefore, the invention includes, in addition to the steps of any method described herein, a step of identifying a subject suffering from fluorosis, dental caries, dentin hypersensitivity or dental calculus, a white spot lesion; a fluorotic lesion; a caries lesion; or an injury caused by dental erosion, or dental plaque or gingivitis or periodontitis.

The present invention provides a composition for mineralizing a tooth surface or subsurface that is also capable of reducing plaque, gingivitis and periodontitis comprising (a) an oral care formulation that includes an ACP and/or ACFP stabilized with associated phosphopeptide to tin, or (b) an ACP or ACFP stabilized with tin-associated phosphopeptide.

In a further aspect, there is provided (a) an oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP and/or ACFP, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP for use in a method of treating or preventing one or more of each of dental cavities, dental cavity, dental erosion and fluorosis, thereby treating or preventing one or more of each of dental cavities, dental cavity, dental erosion and fluorosis. Topical administration of the complex is preferred. The method preferably includes administration of the complex in a formulation as described above.

The present invention also provides a composition containing a tin-associated phosphopeptide-stabilized ACP or ACFP. Preferably, the composition further includes a pharmaceutically acceptable carrier, diluent or excipient.

In a preferred embodiment, the phosphopeptide-stabilized amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) complex in the composition has strongly bound and weakly bound calcium, wherein the bound calcium in the complex is less than calcium tightly bound in an ACP or ACFP complex formed at a pH of 7.0. Optionally, the ACP or ACFP is predominantly presented in a basic form.

In another preferred embodiment, the calcium ion content of the phosphopeptide-stabilized ACP or ACFP complex in the composition is in the range of about 30 to 100 moles of calcium per mole of PP. More preferably, the calcium ion content is in the range of about 30 to about 50 moles of calcium per mole of PP.

In any embodiment as described herein, the ACP and/or ACFP in the composition may be in the form of a complex of ACP and/or ACFP stabilized with casein phosphopeptide.

The invention also relates to a kit for the treatment or prevention of one or more of dental caries, fluorosis, dental hypersensitivity and dental erosion that includes:

(a) a compound capable of increasing or maintaining the pH of a solution, and

(b) an oral care formulation that includes a stannous phosphopeptide-stabilized ACP and/or ACFP, or

(c) a tin-associated phosphopeptide-stabilized ACP or ACFP.

Preferably, the tin-associated phosphopeptide-stabilized ACP or ACFP is in a pharmaceutically acceptable carrier. Desirably, the kit further includes instructions for its use for mineralization of a tooth surface in a patient in need of such treatment. The instructions may describe the use of the kit to treat or prevent one or more of each of dental cavities, tooth loss, dental erosion, dental hypersensitivity, and fluorosis. In one embodiment, the agent and the complex are present in amounts suitable for the treatment of a patient.

The composition or kit of the invention may further include a source of fluoride ions. The fluoride ions may be from any suitable source. A source of fluoride ions may include free fluoride ions or fluoride salts. Examples of sources of fluoride ions include, but are not limited to, the following: sodium fluoride, sodium monofluorophosphate, stannous fluoride, sodium silicofluoride, and amine fluoride. These can be provided in solution (usually an aqueous solution) or in suspension.

Brief description of the drawings

Figure 1: Representative SEM images of subsurface enamel lesions before (A) and after (B) treatment with SnF<2>/stabilized ACP that also demonstrates the superficial layer.

Figure 2: Representative SEM images of subsurface enamel lesions before (A) and after (B) treatment with stabilized NaF/ACP [Note that a new surface layer is not formed].

Figure 3: Scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) of the mineralized surface layer resulting from treatment with stabilized SnF<2>/ACP.

Figure 4: Representative SEM image of the elemental analysis by SEM-EDS of the mineralized surface layer resulting from the treatment with SnF<2>/stabilized ACP.

Figure 5: Representative SEM image of the elemental analysis by SEM-EDS of the mineralized surface layer resulting from the treatment with SnF<2>/stabilized ACP.

Figure 6: Remineralization of subsurface enamel lesions using SnF<2>/stabilized ACP versus NaF/stabilized ACP. The numerical values of this Figure are shown in Table 2.

Figure 7: In situ remineralization of subsurface enamel lesions using CPP-ACP SnF<2>/NaF compared to CPP-ACP, SnF<2>and NaF, NaF with or without CPP-ACP.

Detailed description of the achievements.

The invention disclosed and defined in this specification will be understood to extend to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Other aspects of the present invention and other embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described along with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications and equivalents that may be included within the scope of the present invention as defined in the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

For purposes of interpretation of this specification, terms used in the singular will also include the plural and vice versa.

As used herein, except where the context otherwise requires, the term "comprises" and variations of the term, such as "comprising", "comprises" and "understood", are not intended to exclude additives, components, numbers integers or additional steps. As used herein, except where the context requires otherwise, "comprises" and "includes" may be used interchangeably.

The present invention is based on the finding that casein phosphopeptides can stabilize a stannous compound, such as SnF<2>, in an aqueous environment and in the presence of stabilized amorphous calcium phosphate (ACP) and amorphous calcium fluoride phosphate. stabilized (ACFP), these formulations are superior to other forms of fluoride and ACP or stabilized ACFP in remineralizing subsurface enamel lesions. The mineralization of tooth surfaces can be significantly improved by providing a stannous compound during the mineralization process using stabilized ACP and/or stabilized ACFP. In particular, enamel mineralization by stabilized soluble forms of tin-associated ACP complexes and tin-associated ACFP complexes has been found to be increased compared to stabilized ACP and fluoride without associated tin.

Stannous ion in the presence of stabilized ACP and/or stabilized ACFP can produce a protective surface layer on enamel/dentin to help prevent dental caries, dentin hypersensitivity, dental plaque and periodontal disease. The layer including stannous ions and stabilized ACP and/or stabilized ACFP can provide a reservoir of calcium, phosphate and fluoride for mineralization of the surface or subsurface of the tooth. These formulations have been called tin-associated phosphopeptide-stabilized ACP and tin-associated phosphopeptide-stabilized ACFP.

Without being bound by any theory or mode of action, it is believed that stannous ions are stabilized by the presence of phosphopeptides, particularly casein phosphopeptides. Therefore, phosphopeptides are believed to release stannous and fluoride ions along with calcium and phosphate ions to promote remineralization. Remineralization may be the result of the formation of calcium and stannous fluorapatite. It is also believed that stannous ions cross-link phosphopeptide-stabilized ACP or stabilized ACFP on the tooth surface to form a layer that can protect the tooth surface from demineralization. The presence of tin in the surface layer would make it hard and resistant to degradation due to normal wear or other processes such as erosion. An additional advantage of the invention is that stannous ions can help kill oral bacteria that produce acid and other metabolic products that promote demineralization.

Tin is known to form precipitates with hydroxide ions and phosphate ions, thereby reducing the bioavailability and activity of the stannous ion. The resulting promotion of remineralization driven by stabilized ACP/stannous ion-stabilized ACFP is surprising, as it would be expected that hydroxide ions (e.g., OH-) and phosphate ions (e.g., PO<4> 3-) provided by the stabilized ACP or the stabilized ACFP would cause the precipitation of tin into stannous hydroxide and stannous phosphate, thus severely decreasing any activity of the tin-containing compound. Furthermore, the formation of a precipitate of stannous ions with hydroxide ions would be expected to not only reduce the bioavailability of stannous ions, but also remove hydroxide ions from the environment, thereby lowering the pH. A reduction in pH would promote demineralization and hinder mineralization.

The present invention provides complexes of ACP or ACFP stabilized with tin-associated phosphopeptides and methods for mineralizing a tooth surface or subsurface comprising contacting the tooth surface or subsurface with complexes of ACP or ACFP stabilized with tin-associated phosphopeptides. A tooth subsurface is typically a hypomineralized lesion such that tin-associated phosphopeptide-stabilized ACP or ACFP complexes in contact with the tooth surface migrate through any surface layer, i.e., film and/or plaque, across the surface. porous tooth to the region requiring mineralization. These tin-associated phosphopeptide-stabilized ACP or ACFP complexes also generate a protective surface layer on enamel/dentin to help prevent dental caries, dentin hypersensitivity, dental plaque, and periodontal disease. The tooth surface is preferably dental enamel or dentin. The tooth surface may be a lesion in the enamel, such as a lesion caused by caries, dental erosion or fluorosis.

In one aspect, the invention provides a tin-associated phosphopeptide-stabilized amorphous calcium phosphate (ACP) or a tin-associated amorphous calcium fluoride phosphate (ACFP). The tin may be attached to the phosphopeptide-stabilized amorphous calcium phosphate (ACP) and/or to the amorphous calcium fluoride phosphate (ACFP) as determined using the experimental protocol in Example 2. In one embodiment, the phosphopeptide-stabilized ACP tin-associated or tin-associated phosphopeptide-stabilized ACFP are produced by a method as described herein, including, but not limited to, the method described in Example 1.

Tin is present in the tin-associated phosphopeptide-stabilized amorphous calcium phosphate (ACP) complex or in the tin-associated phosphopeptide amorphous calcium fluoride phosphate (ACFP) complex by binding to or incorporating into the complex. This complex-associated tin, where the tin is bound or incorporated into the complex, can be determined using filtration and atomic absorption spectrophotometry. Tin in the tightly bound complex is measured as the difference between the total tin minus the weakly bound tin. To determine the total tin (both strongly and weakly bound) present in a solution of tin-associated complexes, a solution containing the complexes was taken and placed in HNO3 and incubated at room temperature with slow and constant mixing from end to end. another for 24 hours. The mixture can then be centrifuged at approximately 1,000 g, preferably for 15 minutes at room temperature. The supernatant, when analyzed for stannous ion content, preferably by atomic absorption spectrophotometry (AAS), will provide the value of the total tin present (either bound/associated with the complex or free in solution). The level of weakly bound tin in the solution can be determined by taking a sample of a solution of tin-associated complexes, placing it in a centrifuge with a 1,000 MWCO filter, and centrifuging at approximately 3,000 g, preferably for 1 hour at room temperature, to produce sufficient filtrate (<10% of total sample so as not to affect equilibrium) for analysis by AAS. The filtrates contain weakly bound stannous ions. Strongly bound stannous ions in CPP (colloidal retentate) can be calculated from the difference between total and weakly bound stannous ions. "Weakly bound" tin is tin that can be separated from a complex only by a method such as centrifugation, as described above. Weakly bound tin is still associated with the complexes, but is more easily dissociable than strongly bound stannous ions because of the less stringent conditions explained above.

In one embodiment, the tin-associated phosphopeptide-stabilized amorphous calcium phosphate (ACP) complex or tin-associated amorphous calcium fluoride phosphate (ACFP) complex that includes tin that cannot be separated from the complex by centrifugation at about 3,000 g, preferably for 1 hour at room temperature.

In a further aspect of the present invention there is provided (a) an oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP or ACFP, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP for use in a method of mineralization of a tooth surface comprising providing an ACP or ACFP stabilized with tin-associated phosphopeptide. In a preferred embodiment, the tooth surface is enamel.

In a further aspect of the present invention there is provided (a) an oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP or ACFP, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP for use in a method of treating fluorosis comprising contacting a fluorotic lesion in dental enamel with a tin-associated phosphopeptide-stabilized ACP or ACFP.

In a further aspect of the present invention there is provided (a) an oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP or ACFP, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP for use in a method for treating dental caries comprising contacting a carious lesion in the dental enamel with an ACP or ACFP stabilized with tin-associated phosphopeptide.

In a further aspect of the present invention there is provided (a) an oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP or ACFP, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP for use in a method for the treatment of dental erosion comprising contacting a lesion in the dental enamel caused by erosion with an ACP or ACFP stabilized with tin-associated phosphopeptide.

In a further aspect of the present invention there is provided (a) an oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP or ACFP, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP for use in a method for inhibiting the progression of dental erosion that comprises contacting a surface that presents erosion with an ACP or ACFP stabilized with tin-associated phosphopeptide, thus forming a surface layer that inhibits the progression of dental erosion.

In a further aspect of the present invention there is provided (a) an oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP or ACFP, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP for use in a method of reducing dentin hypersensitivity in exposed dentin comprising contacting the dentin with a tin-associated phosphopeptide-stabilized ACP or ACFP.

In a further aspect of the present invention there is provided (a) an oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP or ACFP, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP for use in a method for remineralizing a lesion in dental enamel comprising contacting the lesion with an ACP or ACFP stabilized with tin-associated phosphopeptide.

In a further aspect of the present invention, an oral care formulation having ACP and/or ACFP stabilized with tin-associated phosphopeptide is provided for use in a method of increasing the effectiveness of remineralization, the method including the step of add a stannous compound to the composition. Preferably, the tin-associated phosphopeptide is added to the composition when it is in an aqueous state, such as during manufacturing. Tin-associated stabilized ACP or ACFP is stabilized by a phosphopeptide. In a preferred embodiment, the phosphopeptide is a casein phosphopeptide. Preferably, the ACP or ACFP is in the form of a complex of ACP or ACFP stabilized with casein phosphopeptide.

In any aspect or embodiments as described herein, the stabilized ACP and/or ACFP may be in a formulation with additional calcium phosphate. Typically, the formulation includes a PP-stabilized ACP and/or ACFP complex together with at least an equal amount by weight of calcium phosphate.

An oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP or ACFP can be contacted with the tooth surface for a period of about 1 to 60 minutes, or for about 1 to 30 minutes. In one embodiment, the tin-associated phosphopeptide-stabilized ACP or ACFP is contacted with the tooth surface for approximately 20 minutes. An example of how this is achieved is to formulate the tin-associated phosphopeptide-stabilized ACP or ACFP into an oral care formulation, such as a paste, and then contact or apply the composition to the tooth surface. The oral care formulation, such as a paste, has sufficient viscosity to be retained on the tooth for the required period of time.

Preferably, a tin-associated phosphopeptide-stabilized ACP or ACFP is contacted with the tooth surface for a period of about 1 minute to 2 hours, or 5 minutes to 60 minutes, or about 10 minutes. Tin-associated phosphopeptide-stabilized ACP or ACFP can be applied repeatedly to the tooth surface over a period of 1 day to several months.

In one embodiment, the tooth surface needs such treatment. Therefore, in another aspect, the invention includes in addition to the steps of any method described herein a step of identifying a subject suffering from fluorosis, dental caries, dentin hypersensitivity or dental calculus, a white spot lesion; a fluorotic lesion; a caries lesion; or an injury caused by dental erosion, dental plaque, gingivitis or periodontitis.

The present invention provides (a) an oral care formulation that includes a tin-associated phosphopeptide-stabilized ACP or ACFP, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP for use in the mineralization of a surface or tooth subsurface and to reduce the viability of bacteria on that tooth surface.

A tin-associated phosphopeptide-stabilized ACP or ACFP complex as mentioned herein includes a tin-associated phosphopeptide-stabilized ACP or ACFP complex formed at a pH of less than 7.0. Preferably, the complex is formed at a pH in the range of about 5.0 to, but below, 7.0. More preferably, the complex is formed in a pH range of about 4.0 to 6.5 or 5.0 to about 6.0. In one embodiment, the pH during formation is maintained at pH 6.5 or less. In a preferred embodiment, the complex is formed at a pH of about 5.5. Preferably, the ACP or ACFP in the complex is predominantly in basic form. The tin-associated phosphopeptide-stabilized ACP or ACFP complex, when produced on an industrial scale, is produced in a bulk solution having a pH greater than about 7.0, preferably about 9.0, however, the local pH in the formation of the complexes it is below about 7.0, preferably about 4.0 to 6.5, preferably about 5.5.

When tin-associated phosphopeptide-stabilized ACP or ACFP, or stabilized ACP or ACFP, is produced in the laboratory in smaller quantities than commercial production, the pH of the entire solution can be maintained at a given pH, that is, if prepared the CPP-ACP at pH 5.5, then all the solution during the formation of CPP-ACP was kept at pH 5.5. However, it may not be necessary or desirable to reduce the pH of the entire bulk solution in commercial manufacturing to 5.5, since the only part of the bulk solution that is required to have the acidic pH is where the complex and mass solution can have. and has localized fluctuations in pH. pH fluctuations arise particularly from the protons provided by the phosphate compound, for example dihydrogen phosphate, as it is added and from the protons released from the acidic phosphate ions as they are converted to the basic form, PO<4>3 -. Therefore, although the overall pH of the bulk solution may be above 7.0, for example about pH 9, the localized pH at which CPP-ACP forms is lower, typically below 7.0. or 6.5, preferably about 4.0 to 6.5, more preferably about 5.5. These fluctuations are localized due to the size of the bulk solution.

A method for forming a tin-associated phosphopeptide-stabilized ACP of the invention is a method comprising the steps of:

(i) obtaining a solution comprising at least one phosphopeptide and;

(ii) mixing solutions comprising calcium ions, phosphate ions and hydroxide ions, while maintaining the pH at about 7.0 or less; and

(iii) mixing a stannous compound;

either

(i) provide a stabilized ACP solution; and

(ii) mix a stannous compound.

A method for forming a stabilized ACFP associated with tin is a method comprising the steps of:

(i) obtaining a solution comprising at least one phosphopeptide and;

(ii) mixing solutions comprising calcium ions, phosphate ions, hydroxide ions and fluoride ions, while maintaining the pH at about 7.0 or less; and

(iii) mixing a stannous compound;

either

(i) provide a stabilized ACFP solution; and

(ii) mix a stannous compound.

Hydroxide ions can be titrated into the solution to maintain the phosphopeptide solution at an essentially constant pH. Calcium and phosphate ions can be titrated into the phosphopeptide solution with constant mixing and at a rate that prevents the formation of a calcium phosphate precipitate in the phosphopeptide solution.

A tin-associated phosphopeptide-stabilized ACP can be produced by a method comprising the step of mixing CPP-ACP and a stannous compound in an aqueous solution, while maintaining the pH at about 6.5 or less.

A tin-associated phosphopeptide-stabilized ACFP can be produced by a method comprising the step of mixing CPP-ACFP and a stannous compound in an aqueous solution, while maintaining the pH at about 6.5 or less.

A tin-associated phosphopeptide-stabilized ACP can be produced by a method comprising the steps of:

(i) obtaining a solution comprising CPP-ACP; and;

(ii) mixing a stannous compound, while maintaining the pH at about 6.5 or less.

A stannous phosphopeptide-stabilized ACFP can be produced by a method comprising the steps of:

(i) obtaining a solution comprising CPP-ACFP; and;

(ii) mixing a stannous compound, while maintaining the pH at about 6.5 or less.

Preferably, the stannous compound is stannous fluoride. Optionally, methods for producing tin-associated phosphopeptide-stabilized ACP or tin-associated phosphopeptide-stabilized ACFP further comprise mixing sodium fluoride in step (ii).

Preferably the pH is maintained with an acid, such as HCl.

Preferably, the solution comprising CPP-ACP or CPP-ACFP is prepared by adding CPP-ACP or CPP-ACFP to distilled or deionized water.

An amorphous calcium phosphate (ACP) complex stabilized with tin-associated phosphopeptide and/or an amorphous calcium fluoride phosphate (ACFP) complex can be formed by mixing a stabilized amorphous calcium phosphate (ACP) complex and/or a amorphous calcium fluoride phosphate (ACFP) complex with stannous fluoride.

A stabilized ACP or ACFP complex as described herein may be the "closed" complexes shown in Figure 2 of Crosset al., 2007.

A stabilized ACP or ACFP complex as mentioned herein includes a stabilized ACP or ACFP complex as described in PCT/AU2005/001781.

In a preferred embodiment, the phosphopeptide-stabilized amorphous calcium phosphate (ACP) complex or amorphous calcium fluoride phosphate (ACFP) has strongly bound and weakly bound calcium, wherein the bound calcium in the complex is less than the strongly bound calcium. bound into an ACP or ACFP complex formed at a pH of 7.0. Optionally, the ACP or ACFP is predominantly presented in a basic form.

A stabilized ACP or ACFP complex as mentioned herein includes a stabilized ACP or ACFP complex formed at a pH less than 7.0. Preferably, the complex is formed at a pH in the range of about 5.0 to, but below, 7.0. More preferably, the complex is formed in a pH range of about 5.0 to about 6.0. In a preferred embodiment, the complex is formed at a pH of about 5.5. Preferably, the ACP or ACFP in the complex is predominantly in basic form.

A stabilized ACP can be produced by a method comprising the steps of:

(i) obtaining a solution comprising at least one phosphopeptide and;

(ii) mixing solutions comprising calcium ions, phosphate ions and hydroxide ions, while maintaining the pH at about 7.0 or less.

A stabilized ACFP can be produced by a method comprising the steps of:

(i) obtaining a solution comprising at least one phosphopeptide and;

(ii) mixing solutions comprising calcium ions, phosphate ions, hydroxide ions and fluoride ions, while maintaining the pH at about 7.0 or less.

A phosphopeptide-stabilized amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) complex may also include wherein the ACP in the complex has strongly and weakly bound calcium, wherein the strongly bound calcium in the complex is less than the tightly bound calcium in an ACP or ACFP complex formed at a pH of 7.0 and the ACP or ACFP is predominantly in basic form, obtainable or obtained by a method comprising:

a) mixing a first solution comprising calcium ions, a second solution comprising phosphate ions and optionally a third solution comprising fluoride ions, to a solution comprising phosphopeptides and a solvent with a pH of about 5 up to, but below 7 ; and

b) maintain the pH of the solution at approximately 5.0 up to, but below 7.0 during mixing by adding hydroxide ions.

"Strongly" and "weakly" bound calcium and phosphate in ACP or ACFP can be determined by analytical ultrafiltration. Briefly, the solution of phosphopeptide, calcium, phosphate and optionally fluoride, mixed while maintaining the pH at about 7.0 or less can be first filtered through a 0.1 micron filter to remove free calcium and phosphate that They are not associated with complexes. This free calcium and phosphate are present in the filtrate and are discarded. Any free calcium or phosphate that is not associated in any way with the complexes would not be bioavailable, that is, it would not be delivered by the phosphopeptide to the tooth. The retentate from the 0.1 micrometer filtration can be further analyzed by centrifugation through a 3,000 mw cutoff filter at 1,000 g for 15 min. The resulting filtrate contains calcium and phosphate that are weakly bound or associated with the complexes. Thanks to this centrifugal force, calcium and phosphate that are not tightly bound to the complexes are released and enter the filtrate. Ca and Pi that are tightly bound in the complexes are retained in the retentate. The amount of tightly bound Ca and Pi in the retentate can be determined by subtracting the amount of Ca and Pi in the filtrate from the total amount of Ca and Pi in the retentate of the 0.1 micrometer filtrate.

A stabilized ACP or ACFP complex as mentioned herein includes a stabilized ACP or ACFP complex as described in PCT/AU2006/000885.

An amorphous calcium phosphate (ACP) complex or amorphous calcium phosphate fluoride (ACFP) complex stabilized with "supercharged" phosphopeptide or phosphoprotein (PP). The complex can form at any pH (eg, 3-10). Preferably, the phosphopeptide includes the sequence -A-B-C-, where A is a phosphoamino acid, preferably phosphoserine, B is any amino acid including a phosphoamino acid and C is glutamic acid, aspartic acid or a phosphoamino acid. The phosphoamino acid may be phosphoserine. PP is overloaded with calcium and phosphate ions. Calcium ions can be in the range of 30-1,000 moles of Ca per mole of PP, or in the range of 30-100 or 30-50 moles of Ca per mole of PP. In another embodiment, the mole of Ca per mole of PP is at least 25, 30, 35, 40, 45 or 50.

The complex of amorphous calcium phosphate or amorphous calcium fluoride phosphate stabilized with phosphopeptide or phosphoprotein (PP) may have a calcium ion content greater than about 30 moles of calcium per mole of PP. In a preferred embodiment, the calcium ion content is in the range of about 30 to 100 moles of calcium per mole of PP. More preferably, the calcium ion content is in the range of about 30 to about 50 moles of calcium per mole of PP.

The amorphous calcium phosphate (ACP) complex or amorphous calcium phosphate fluoride (ACFP) complex stabilized with phosphopeptide or phosphoprotein (PP) can be produced by a method comprising the steps of:

(i) obtain solutions comprising calcium, inorganic phosphate and fluoride (optional); and

(ii) mix (i) with a solution comprising PP-ACP.

In a preferred embodiment, the PP is casein phosphopeptide (CPP).

The PP-stabilized ACP and/or ACFP complex may further include at least an equal amount by weight of calcium phosphate. Preferably, the calcium phosphate is CaHPO4. Preferably, the calcium phosphate (e.g., CaHPO4) is dry mixed with the PP-stabilized ACP and/or ACFP complex. In a preferred embodiment, the ratio of the PP-ACP and/or PP-ACFP complex: calcium phosphate is approximately 1:1-50. more preferably about 1:1-25, more preferably about 1:5-15. In one embodiment, the ratio of the PP-ACP and/or PP-ACFP complex: calcium phosphate is approximately 1:10.

The oral care formulation including a complex of amorphous calcium phosphate (ACP) stabilized with phosphopeptide or phosphoprotein (PP) and/or amorphous calcium fluoride phosphate (ACFP) having a calcium ion content greater than about 30 moles of calcium per mole of PP when used in the oral cavity can be produced by a method that includes the steps of:

(i) obtaining a powder that includes a PP-ACP and/or PP-ACFP complex;

(ii) dry mixing with an effective amount of calcium phosphate; and

(iii) formulating the dry mixture of PP-ACP and/or PP-ACFP and calcium phosphate into an oral care formulation.

Preferably, the form of calcium phosphate for dry mixing is any soluble calcium phosphate including, but not limited to, CaHPO4, Ca2HPO4 and calcium lactate.

The present invention also provides a tin-associated phosphopeptide-stabilized ACP or ACFP for use in a method of mineralization of a tooth surface or subsurface that includes the steps of:

(i) contact the tooth surface with a protein disrupting agent, and

(ii) contacting the tooth surface with (a) an oral care formulation that includes an ACP and/or ACFP stabilized with tin-associated phosphopeptide, or (b) an ACP or ACFP stabilized with tin-associated phosphopeptide.

The tooth surface is preferably tooth enamel. In one embodiment, the tooth surface is a lesion in the enamel, such as a lesion caused by caries, dental erosion or fluorosis. Any suitable protein disrupting agent can be used in the method of the present invention. The agent is required to reduce the protein barrier formed on the surface to be treated, such as the film on the teeth. Examples of suitable agents include bleach, detergent, chaotropic agents such as urea, high concentrations of phosphate, protease cocktails (e.g., endopeptidases, proteinases and exopeptidases) and any other protein solubilizing, disrupting or hydrolyzing agents. Examples of suitable bleaches include sodium hypochlorite (NaOCI) and cabamide peroxide bleaches. In a preferred embodiment, the bleach is an alkaline bleach. In another preferred embodiment, the alkaline bleach is NaOCI. The protein disrupting agent acts to solubilize and partially or completely remove tooth surface proteins, particularly pellicle proteins.

A composition as described herein may further include free fluoride ions. The fluoride ions may be from any suitable source. A source of fluoride ions may include free fluoride ions or fluoride salts. Examples of sources of fluoride ions include, but are not limited to, the following: sodium fluoride, sodium monofluorophosphate, stannous fluoride, sodium silicofluoride, and amine fluoride. These can be provided in solution (usually an aqueous solution) or in suspension.

Fluoride ions are preferably present in the composition in an amount greater than 1 ppm. More preferably, the amount is greater than 3 ppm. In another embodiment, it is preferably greater than 10 ppm. In typical embodiments described below, the amount may be several hundred or thousands of ppm. Fluoride content is typically measured as ppm in oral compositions in the manner commonly used in the art. When fluoride comes from a source with stabilized ACP, ppm refers to the concentration of fluoride in that source, typically a solution or suspension of bioavailable fluoride.

"Phosphopeptide" in the context of the description of this invention means an amino acid sequence wherein at least one amino acid is phosphorylated. Preferably, the phosphopeptide includes one or more of the amino acid sequence -A-B-C-, where A is a phosphoamino residue , B is any amino acyl residue that includes a phosphoamino residue and C is selected from a glutamyl, aspartyl or phosphoamino residue. Any of the phosphoamino residues may independently be a phosphoseryl residue. B is desirably a residue whose side chain is neither relatively large. nor hydrophobic. It can be Gly, Ala, Val, Met, Leu, Ile, Ser, Thr, Cys, Asp, Glu, Asn, Gln or Lys.

In another embodiment, at least two of the phosphoamino acids in the sequence are preferably contiguous. Preferably the phosphopeptide includes the sequence A-B-C-D-E, where A, B, C, D and E are independently phosphoserine, phosphothreonine, phosphotyrosine, phosphohistidine, glutamic acid or aspartic acid, and at least two, preferably three, of A, B, C, D and E are a phosphoamino acid. In a preferred embodiment, the phosphoamino acid residues are phosphoserine, most preferably three contiguous phosphoserine residues. It is also preferred that D and E are independently glutamic or aspartic acid.

In one embodiment, the ACP or ACFP is stabilized by a casein phosphopeptide (CPP), which is in the form of intact casein or casein fragment, and the complex formed preferably has the formula [CPP(ACP)8]n or [(CPP )(ACFP)8]n where n is equal to or greater than 1, e.g. 6. The complex formed may be a colloidal complex, where the core particles aggregate to form large colloidal particles (e.g. 100 nm ) suspended in water. Thus, PP can be a protein or a casein phosphopeptide.

The PP can be from any source; It may be present in the context of a larger polypeptide, including a full-length casein polypeptide, or it may be isolated by tryptic digestion or other enzymatic or chemical digestion of casein, or other phosphoamino acid-rich proteins such as phosphitin, or by chemical synthesis. or recombinant, provided it comprises the sequence -A-B-C- or A-B-C-D-E as described above. The sequence flanking this central sequence can be any sequence. However, those flanking sequences in as1(59-79) [1], (3(1-25) [2], as2(46-70) [3] and as2(1-21) [4] are preferred. The flanking sequences may optionally be modified by conservative deletion, addition or substitution of one or more residues. The amino acid composition and sequence of the flanking region are not critical.

Examples of conservative substitutions are shown in Table 1 below.

Table 1

Flanking sequences may also include amino acid residues that do not occur naturally. Commonly found amino acids that are not encoded by the genetic code include:

2-amino adipic acid (Aad) for Glu and Asp;

2-aminopimelic acid (Apm) for Glu and Asp;

2-aminobutyric acid (Abu) for Met, Leu and other aliphatic amino acids;

2-aminoheptanoic acid (Ahe) for Met, Leu and other aliphatic amino acids;

2-aminoisobutyric acid (Aib) for Gly;

cyclohexylalanine (Cha) for Val, Leu and Ile;

homoarginine (Har) for Arg and Lys;

2,3-diaminopropionic acid (Dpr) for Lys, Arg and His;

N-ethylglycine (EtGly) for Gly, Pro and Ala;

N-ethylasparygin (EtAsn) for Asn and Gln;

Hydroxylysine (Hyl) for Lys;

allohydroxylysine (AHyl) for Lys;

3-(and 4)hydroxyproline (3Hyp, 4Hyp) for Pro, Ser and Thr;

aloisoleucine (Alle) for Ile, Leu and Val;

p-amidinophenylalanine for Ala;

N-methylglycine (MeGly, sarcosine) for Gly, Pro, Ala.

N-methylisoleucine (Melle) for Ile;

Norvaline (Nva) for Met and other aliphatic amino acids;

Norleucine (Nle) for Met and other aliphatic amino acids;

Ornithine (Orn) for Lys, Arg and His;

Citrulline (Cit) and methionine sulfoxide (MSO) for Thr, Asn, and Gln;

N-methylphenylalanine (MePhe), trimethylphenylalanine, halo (F, Cl, Br and I) phenylalanine, trifluorylphenylalanine, for Phe.

In one embodiment, the PP is one or more phosphopeptides selected from the group consisting of as1(59-79) [1], (3(1-25) [2], as2(46-70) [3] and as2( 1-21) [4]:

[1] Gln59-Met-Glu-Ala-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ile-Val-Pro-Asn-Ser(P )-Val-Glu-Gln-Lys79 as1(59-79)

[2] Arg1-Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-Gly-Glu-Ile-Val-Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser( P)-Glu -Glu-Ser-Ile-Thr-Arg253(1-25)

[3] Asn46-Ala-Asn-Glu-Glu-Glu-Tyr-Ser-Ile-Gly-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser(P)-Ala-Glu -Val-Ala-Thr-Glu-Glu-Val-Lys70 as2(46-70)

[4] Lys1-Asn-Thr-Met-Glu-His-Val-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser-Ile-Ile-Ser(P)-Gln-Glu -Thr-Tyr-Lys21 as2(1-21).

In another embodiment of the invention, the tin-associated phosphopeptide-stabilized ACP complex and/or tin-associated phosphopeptide-stabilized ACFP is incorporated into oral compositions such as toothpaste, mouthwashes or oral formulations to aid in prevention. and/or the treatment of dental caries, tooth loss, dental erosion or fluorosis, dentin hypersensitivity, dental plaque, gingivitis or periodontitis. Oral compositions comprising an amount of ACP and/or ACFP stabilized with tin-associated phosphopeptide sufficient to form a layer on a tooth surface, preferably the layer having a calcium:phosphate ratio equivalent to normal apatite, for example, the ratio It is approximately 2:1. The layer may contain an amount of calcium of approximately 20% by weight. Preferably, the layer may have approximately an elemental analysis of any of the elements, such as calcium, phosphate, fluoride, carbon and/or tin, as shown in any one of Tables 3 or 4. The ACP and/or ACFP complexes stabilized with tin-associated phosphopeptide may comprise 0.01-50% by weight of the composition, preferably 1.0-50%. For oral compositions, it is preferred that the amount of tin-associated phosphopeptide-stabilized ACP or ACFP complexes administered be 0.01-50% by weight, preferably 1.0%-50% by weight of the composition. In a particularly preferred embodiment, the oral composition of the present invention contains approximately 2% ACP or ACFP complexes stabilized with tin-associated phosphopeptides or a mixture of both. The oral composition of this invention containing the aforementioned agents can be prepared and used in various forms applicable to the mouth such as dentifrices including toothpastes, dental powders and liquid dentifrices, mouth rinses, mouthwashes, oral sprays, varnishes, dental cement , lozenges, chewing gum, toothpastes, gingival massage creams, gargle tablets, dairy products and other food products. The oral composition according to this invention may further include additional well-known ingredients depending on the type and form of a particular oral composition. Certain compositions of the invention such as toothpastes, tooth powders and liquid dentifrices, mouth rinses, mouth washes and oral sprays have a relatively low viscosity and have a remineralizing effect without a significant residence time in the oral cavity.

In certain preferred forms of the invention, an oral composition may be substantially liquid in character, such as a mouthwash, wash or spray. In such a preparation, the carrier is typically a mixture of water and alcohol desirably including a humectant as described below. Generally, the weight ratio of water to alcohol is in the range of about 1:1 to about 20:1. The total amount of water and alcohol mixture in this type of preparation is typically in the range of about 70 to about 99.9% by weight of the preparation. The alcohol is typically ethanol or isopropanol. Ethanol is preferred.

In other desirable forms of this invention, the composition may be of substantially solid or pasty character, such as tooth powder, a dental tablet or a toothpaste (toothpaste) or toothpaste gel. The vehicle of such solid or pasty oral preparations generally contains a dentally acceptable polishing material. Examples of polishing materials are water-insoluble sodium metaphosphate, potassium metaphosphate, tricalcium phosphate, calcium phosphate dihydrate, dicalcium phosphate anhydrous, calcium pyrophosphate, magnesium orthophosphate, trimagnesium phosphate, calcium carbonate, hydrated alumina, alumina calcined, aluminum silicate, zirconium silicate, silica, bentonite and mixtures thereof. Other suitable polishing materials include particulate thermosetting resins such as melamine formaldehydes, phenolics and urea, and cross-linked polyepoxides and polyesters. Preferred polishing materials include crystalline silica having particle sizes of up to about 5 micrometers, an average particle size of up to about 1.1 micrometers and a surface area of up to about 50,000 cm2/g., silica gel or colloidal silica. and complex amorphous alkali metal aluminosilicate.

When using visually clear gels, a colloidal silica polishing agent, such as those sold under the SYLOID brand name as Syloid 72 and Syloid 74 or under the SANTOCEL brand name as Santocel 100, alkali metal aluminosilicate complexes are particularly useful as they have indices nearby refraction. to the refractive indices of gelling agent-liquid systems (including water and/or humectant) commonly used in dentifrices.

Many of the so-called "water-insoluble" polishing materials are anionic in character and also include small amounts of soluble material. Thus, insoluble sodium metaphosphate can be formed in any suitable manner, for example, as illustrated in Thorpe's Dictionary of Applied Chemistry, Volume 9, 4th Edition, p. 510 511. The forms of insoluble sodium metaphosphate known as Madrell salt and Kurrol salt are other examples of suitable materials. These metaphosphate salts exhibit only minimal solubility in water and are therefore commonly referred to as insoluble metaphosphates (IMPs). A minor amount of soluble phosphate material is present in these as impurities, usually a small percentage, such as up to 4% by weight. The amount of soluble phosphate material, which is believed to include a soluble sodium trimetaphosphate in the case of insoluble metaphosphate, can be reduced or removed by washing with water, if desired. Insoluble alkali metal metaphosphate is normally used in powder form with a particle size such that no more than 1% of the material is larger than 37 micrometers.

The polishing material is generally present in the solid or pasty compositions in concentrations by weight of about 10% to about 99%. Preferably, it is present in amounts of about 10% to about 75% in the toothpaste, and about 70% to about 99% in the tooth powder. In toothpastes, when the polishing material is silica in nature, it is generally present in an amount of about 10-30% by weight. Other polishing materials are typically present in an amount of about 30-75% by weight.

In a toothpaste, the liquid carrier may comprise water and humectant typically in an amount ranging from about 10% to about 80% by weight of the preparation. Glycerin, propylene glycol, sorbitol and polypropylene glycol are examples of suitable humectants/vehicles. Liquid mixtures of water, glycerin and sorbitol are also advantageous. In transparent gels where refractive index is an important consideration, about 2.5-30% w/w water, 0 to about 70% w/w glycerin, and about 20-80% w/w are preferably used. p of sorbitol.

Toothpastes, creams and gels typically contain a natural or synthetic thickener or gelling agent in proportions of about 0.1 to about 10, preferably about 0.5 to about 5% w/w. A suitable thickener is synthetic hectorite, a synthetic colloidal alkali metal magnesium silicate complex clay available, for example, as Laponite (e.g. CP, SP 2002, D) marketed by Laporte Industries Limited. Laponite D is approximately 58.00% by weight SiO<2>, 25.40% MgO, 3.05% Na<2>O, 0.98% Li<2>O, and some water and trace metals. Its true specific gravity is 2.53 and it has a bulk density of 1.0 g/ml at 8% moisture.

Other suitable thickeners include Irish moss, iota carrageenan, gum tragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose, hydroxybutylmethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose (e.g. available as Natrosol), sodium carboxymethylcellulose and colloidal silica such as finely ground Syloid (e.g.

244). Also included may be solubilizing agents such as wetting polyols such as propylene glycol, dipropylene glycol and hexylene glycol, cellosolves such as methyl cellosolve and ethyl cellosolve, vegetable oils and waxes containing at least about 12 carbons in a linear chain such as olive oil, olive oil. castor. and petrolatum and esters such as amyl acetate, ethyl acetate and benzyl benzoate.

It will be understood that, as is conventional, oral preparations will normally be sold or otherwise distributed in appropriately labeled containers. Therefore, a bottle of mouthwash will have a label describing it, in essence, as a mouthwash or mouthwash and with instructions for its use; and a toothpaste, cream or gel will generally be in a collapsible tube, typically aluminum, lead-lined or plastic, or other pressurized dispenser, pump or dispenser for dispensing the contents, which will have a label describing it, in essence, like a toothpaste, gel or toothpaste.

Organic surfactants can be used in the compositions of the present invention to achieve greater prophylactic action, help achieve thorough and complete dispersion of the active agent throughout the oral cavity and make the present compositions more cosmetically acceptable. The organic surfactant material is preferably anionic, nonionic or ampholytic in nature and preferably does not interact with the active agent. It is preferred to use as a surfactant a detergent material that gives the composition detergent and foaming properties. Suitable examples of anionic surfactants are water-soluble salts of monosulfates of monoglycerides of higher fatty acids, such as the sodium salt of the monosulfated monoglyceride of hydrogenated coconut oil fatty acids, higher alkyl sulfates such as sodium lauryl sulfate, alkylaryl sulfonates such as sodium dodecylbenzene sulfonate, higher alkylsulfoacetates, higher fatty acid esters of 1,2-dihydroxypropanesulfonate and the substantially saturated higher aliphatic acylamides of lower aliphatic aminocarboxylic acid compounds, such as those having 12 to 16 carbons in the fatty acid, alkyl or acyl radicals, and the like. Examples of the latter-mentioned amides are N-lauroylsarcosine and the sodium, potassium and ethanolamine salts of N-lauroyl, N-myristoyl or N-palmitoylsarcosine, which should be substantially free of soap or similar higher fatty acid material. The use of these sarconite compounds in the oral compositions of the present invention is particularly advantageous since these materials exhibit a marked prolonged effect in inhibiting acid formation in the oral cavity due to the breakdown of carbohydrates, in addition to exerting some reduction in the solubility of tooth enamel in acidic solutions. Examples of water-soluble nonionic surfactants suitable for use are condensation products of ethylene oxide with various compounds containing reactive hydrogen and having long hydrophobic chains (e.g., aliphatic chains of about 12 to 20 carbon atoms). ), whose condensation products ("ethoxamers") contain hydrophilic polyoxyethylene moieties, such as condensation products of poly(ethylene oxide) with fatty acids, fatty alcohols, fatty amides, polyhydric alcohols (e.g., sorbitan monostearate ) and polypropylene oxide (e.g., pluronic materials).

The surfactant is typically present in an amount of about 0.1-5% by weight. It is noteworthy that the surfactant can help dissolve the active agent of the invention and therefore decrease the amount of solubilizing humectant needed.

Various other materials may be incorporated into the oral preparations of this invention such as bleaching agents, preservatives, silicones, chlorophyll compounds and/or ammoniacal material such as urea, diammonium phosphate and mixtures thereof. These adjuvants, when present, are incorporated into the preparations in amounts that do not substantially adversely affect the desired properties and characteristics.

Any suitable flavoring or sweetening material may also be used. Examples of suitable flavoring constituents are flavoring oils, e.g. e.g., spearmint oil, spearmint, wintergreen, sassafras, clove, sage, eucalyptus, marjoram, cinnamon, lemon and orange, and methyl salicylate. Suitable sweetening agents include sucrose, lactose, maltose, sorbitol, xylitol, sodium cyclamate, perilarthin, AMP (aspartyl phenyl alanine, methyl ester), saccharin and the like. Suitably, the flavoring and sweetening agents may comprise, each or together, from about 0.1% to 5% more of the preparation.

The compositions of this invention may also be incorporated into lozenges, or chewing gum or other products, e.g. e.g., by stirring in a warm rubber base or coating the outer surface of a rubber base, examples of which are jelutong, rubber latex, vinylite resins, etc., desirably with conventional plasticizers or softeners, sugar or other sweeteners or such as glucose, sorbitol and the like. The composition of the invention may be a dual phase composition where each phase allows the release of components for different periods of time. For example, in use, a dual phase composition may release tin-associated phosphopeptide-stabilized ACP and/or tin-associated phosphopeptide-stabilized ACFP, preferably CPP-ACP/SnF<2>and/or CPP-ACFP/SnF<2 >, of a first phase at a faster rate than a compound that is capable of increasing or maintaining the pH of a solution of a second phase. Preferably, the dual phase composition is a dual phase chewing gum.

An alternative composition may be one that provides phosphopeptide-stabilized ACP or ACFP and a stannous compound that then in situ, such as the oral cavity, forms stannous-associated phosphopeptide-stabilized ACP or ACFP. An exemplary composition may be a chewing gum containing stabilized ACP or ACFP in the granule and a stannous compound in the chewable center.

In a further aspect, the invention provides compositions including pharmaceutical compositions comprising any of (a) oral care formulations including a tin-associated phosphopeptide-stabilized ACP and/or ACFP, or (b) complexes of ACP or ACFP. stabilized with tin-associated phosphopeptide as described above together with a compound capable of increasing or maintaining the pH of a solution and a pharmaceutically acceptable carrier. Said compositions can be selected from the group consisting of dental compositions, anticariogenic compositions and therapeutic compositions. Dental compositions or therapeutic compositions may be in the form of a gel, liquid, solid, powder, cream or lozenge. Therapeutic compositions may also be in the form of tablets or capsules. In one embodiment, tin-associated phosphopeptide-stabilized ACP or ACFP complexes are substantially the only remineralizing active components of said composition. For example, a cream formulation containing: water; glycerol; CPP-ACP/SnF<2>; D-sorbitol; silicon dioxide; sodium carboxymethylcellulose (CMC-Na); propylene glycol; titanium dioxide; xylitol; phosphoric acid; guar gum; zinc oxide; sodium saccharin; ethyl phydroxybenzoate; magnesium oxide; butyl p-hydroxybenzoate and propyl p-hydroxybenzoate.

The invention further includes a formulation described above provided together with instructions for its use to treat or prevent any one or more of dental caries or dental decay, dental erosion and fluorosis, dentin hypersensitivity, dental plaque, gingivitis or periodontitis.

In another embodiment, the compositions of the invention as described herein do not include a phosphate buffer and/or a calcium chelator. For example, any dentifrice described herein may not include a phosphate buffer and/or a calcium chelator.

In one embodiment of the present invention there is provided a composition for dental mineralization that includes ACP or ACFP complexes stabilized with tin-associated phosphopeptides, wherein the composition does not include a phosphate buffer and/or a calcium chelator.

In another embodiment, the compositions of the invention as described herein do not include a viscosity regulator, or a 0.5 to 50% viscosity regulator.

In another embodiment, the compositions of the invention as described herein do not include sodium carboxymethyl cellulose, or 0.01 to 10% sodium carboxymethyl cellulose having a degree of esterification of 0.7 to 1.0.

In one embodiment, the active components of the composition consist essentially of ACP or ACFP complexes stabilized with tin-associated phosphopeptides.

In a further aspect, a tin-associated phosphopeptide-stabilized ACP or ACFP complex is provided for use in a method of treating or preventing one or more of each of dental caries, tooth loss, dental erosion and fluorosis, dentin hypersensitivity, dental plaque, gingivitis and periodontitis comprising the steps of administering (a) an oral care formulation that includes an ACP and/or ACFP stabilized with tin-associated phosphopeptide, or (b) a complex of ACP or ACFP stabilized with associated phosphopeptide to tin, to a subject's teeth. Topical administration of the complex is preferred. The method preferably includes administration of the complex in a formulation as described above.

In a further aspect, there is provided the use of (a) oral care formulations that include an ACP and/or ACFP stabilized with tin-associated phosphopeptide, or (b) a complex of ACP or ACFP stabilized with tin-associated phosphopeptide in a manufacture of a composition for the treatment and/or prevention of one or more of dental caries, dental loss, dental erosion and fluorosis, dental hypersensitivity, dental plaque, gingivitis and periodontitis.

According to a further aspect of the invention, there is provided a dental restorative composition, which includes a dental restorative material to which has been added (a) an oral care formulation that includes an ACP and/or ACFP stabilized with associated phosphopeptide to tin, or (b) a complex of ACP or ACFP stabilized with tin-associated phosphopeptide. The base of the dental restorative material may be a glass ionomer cement, a composite material, or any other compatible restorative material. A glass ionomer cement is preferred. It is preferred that the amount of tin-associated phosphopeptide-stabilized ACP or ACFP complexes, preferably CPP-ACP/SnF<2>complex or CPP-ACFP/SnF<2>complex included in the dental restorative material is 0 0.01-80% by weight, preferably 0.5-10% and more preferably 1-5% by weight. The dental restorative material of this invention containing the aforementioned agents can be prepared and used in various forms applicable to dental practice. The dental restorative material according to this embodiment may further include other ions, e.g. e.g., antibacterial ions Zn2+, Ag+, etc. or other additional ingredients depending on the type and form of a particular dental restorative material. It is preferable that the pH of the dental restorative material according to this embodiment is between 2-10, more preferably 5-9 and even more preferably 5-7. It is preferable that the pH of the dental restorative material containing a complex of ACP or ACFP stabilized with tin-associated phosphopeptide is in the range of about 2 to 10, more preferably in the range of about 5 to 9 and even more preferably in the range from about 5 to 7.

It will be clearly understood that, although this specification relates specifically to human applications, the invention is also useful for veterinary purposes. Thus, in all respects, the invention is useful for domestic animals such as cattle, sheep, horses and poultry; for pets such as dogs and cats; and for zoo animals.

An example of a mineralizing composition comprises the following (in decreasing order of proportion):

water

glycerol

CPP-ACP/SnF<2>

D-sorbitol

silicon dioxide

sodium carboxymethylcellulose (CMC-Na)

propylene glycol

titanium dioxide

xylitol

phosphoric acid

guar gum

zinc oxide

sodium saccharin

ethyl p-hydroxybenzoate

magnesium oxide

butyl p-hydroxybenzoate

propyl p-hydroxybenzoate

The invention will now be described in more detail with reference to the following non-limiting examples.

Example 1

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) was purchased from Cadbury Enterprises Pte Ltd under the trade name Recaldent™. A solution was prepared using CPP-ACP, SnF<2>and NaF to produce 0.4% w/v CPP-ACP, 220 ppm F as SnF<2>and 70 ppm F as NaF, pH 5.6 . Specifically, CPP-ACP/SnF<2>complexes were prepared by adding CPP-ACP to distilled/deionized water and then SnF<2>(solid) and NaF were added with the addition of 1 M HCl to maintain the pH between 4. 0 - 6.5. The pH was not allowed to rise above 6.5. The total volume of acid added was less than 1% of the volume of CPP-ACP/SnF<2> solution. This solution was called stabilized SnF<2>/ACP. Although NaF was added in this method, it is a minor component and most of the fluoride is derived from SnF<2>. The method could be performed using SnF<2>only (without NaF). Another solution was prepared using CPP-ACP and NaF to produce 0.4% w/v CPP-ACP and 290 ppm F as NaF, pH 5.6. This solution was called stabilized NaF/ACP.

Both solutions were stable at room temperature (20 °C) for many months without precipitating. The ability of both solutions to remineralize subsurface enamel lesions was tested. Demineralized subsurface lesions of human dental enamel were prepared in third molar enamel blocks using the method of Reynolds, (1997). J Dent Res 76:1587-1595. Half of the blocks were remineralized by suspending them individually in the stabilized SnF<2>/ACP solution and the other half in the stabilized NaF/ACP solution for 7 days at 37 °C. After remineralization, enamel blocks were embedded, sectioned, and subjected to cross-sectional microradiography and densitometric image analysis as previously described by Reynolds (1997) to determine the percent mineral content gain (% remineralization) shown in Table 2. A one-way analysis of variance with differences in means determined using a Tukey HSD post hoc comparison showed that treatment with stabilized SnF<2>/ACP solution significantly increased the level of remineralization by 32%.

TABLE 2. Remineralization of subsurface enamel lesions using stabilized SnF<2>/ACP versus stabilized NaF/ACP.

*significantly different p < 0.01

Analysis of the enamel lesions by scanning electron microscopy (Figures 1 and 2) revealed that the lesions treated with stabilized SnF<2>/ACP also had a superficial layer. Surprisingly, the stabilized SnF<2>/ACP formulation produced significantly greater remineralization of the subsurface enamel lesion (Table 2) and also appeared to form a protective surface layer demonstrating the unexpected superiority of the SnF<2>/ACP formulation. stabilized. An experiment using scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) of the surface layer (Figure 3 and Table 3) confirmed that it was apatite. EDS analysis of the surface layer showed that it contained 10.33 wt% P and 21.18 wt% Ca, giving a typical apatite wt% Ca:P ratio of 2.05. Healthy enamel contained 15.26 wt% P and 30.99 wt% Ca, giving the same apatite-typical Ca:P wt% ratio of 2.03.

These data show that the surface layer is composed of apatite, however, the surface layer had higher Sn (1.23 wt% vs. 0 wt.% for healthy enamel) and fluoride (0.54 wt.% vs. 0.11% for healthy enamel). Of course, this is consistent with the CPP-stabilized SnF<2>/ACP solution containing Sn and F. CPP was also involved in the formation of the surface layer since C was 26.53 wt% in the superficial layer, but only about 10% by weight in healthy enamel (Table 4). This supports the mechanism by which stannous ions can cross-link stabilized ACP or phosphopeptide-stabilized ACFP on the tooth surface to form a layer that can protect the tooth surface from demineralization.

These results indicate that treatment with CPP-stabilized SnF<2>/ACP has not only produced substantially better subsurface remineralization, but has also produced a uniform protective surface layer.

The retention of ACP or ACFP stabilized with tin-associated phosphopeptide in a cross-linked surface layer would (i) protect against dental sensitivity since it would seal the dentinal tubules, (ii) protect against plaque formation since it contains the antibacterial ion Sn and ( iii) it would protect against dental erosion and dental caries. It would also promote subsurface remineralization. Without being tied to any theory or mechanism of action, it appears that stannous ions help promote the surface layer by cross-linking CPP-ACP or CPP-ACFP on the enamel surface.

TABLE3. Elemental analysis by SEM-EDS of the mineralized surface layer resulting from the treatment with stabilized SnF<2>/ACP.

A more detailed scanning electron microscopy - energy dispersive spectroscopy (SEM-EDS) analysis of the various layers where the inclusion resin is excluded is shown in Table 4. This shows that the high carbon content in the healthy enamel in Table 3 includes the resin. When resin was excluded, the carbon content of healthy enamel was in the range of 6 to 10 wt %. The carbon content of the surface layer produced by treatment with CPP-stabilized SnF<2>/ACP was almost 3 times higher than that of healthy enamel, indicating that phosphopeptides are present in the layer. The protective surface layer formed by ACP complexes stabilized with tin-associated phosphopeptides provides a source of fluoride since the fluoride level in the mineralized surface layer is 5 times higher than the fluoride level in healthy enamel. Furthermore, the level of fluoride present is higher than that present in the remineralized lesion. The level of tin present in the superficial layer was significantly higher, 12 times, than the remineralized lesion.

The level of fluoride in the remineralized lesion with ACP associated with tin is higher than CPP-ACP and sodium fluoride. Additionally, there was an increase in calcium present in the remineralized lesion by ACP stabilized with tin-associated phosphopeptide compared to CPP-ACP and sodium fluoride. This calcium level is close to the level of calcium present in healthy enamel.

TABLE 4.Detailed elemental analysis by SEM-EDS of the mineralized surface layer resulting from treatment with SnF<2>/ACP stabilized with CPP.

to. Sabel et al. Scientific World Journal (2012)

b. ND = not yet determined

C. Ratio in % by weight of Ca:P for HA = 2.157 and for ACP = 1.941

Example 2

This example describes the experimental protocol for the measurement of free, CPP-bound (tightly bound), and weakly bound ions in solution.

A sample was taken from each solution prepared in Example 1 and less than 10% was collected as filtrate using a Centriprep 3 ultrafiltration membrane with molecular weight cutoff of 3,000. Centriprep containing samples were centrifuged at 1,000 g for 15 minutes in a Beckman J2-21 centrifuge using a JA 10.5 rotor. The original sample before Centriprep centrifugation and a sample of the filtrate after Centriprep centrifugation were taken to analyze calcium, phosphate fluoride, and tin concentrations. Analysis of the original sample gave total concentrations of calcium, phosphate, fluoride and tin ions and analysis of the filtrate gave concentrations of free (unbound) calcium, phosphate and fluoride. The difference between the total and unbound concentrations is the concentration of Ca, Pi, F, and Sn bound by the CPP.

Example 3

A topical cream can be produced according to the present invention having the following ingredients:

Water

glycerol

SnF<2>/ACP and/or stabilized SnF<2>/ACFP

D-sorbitol

sodium carboxymethylcellulose (CMC-Na)

propylene glycol

silicon dioxide

titanium dioxide

xylitol

phosphoric acid

sodium fluoride

flavoring

sodium saccharin

ethyl p-hydroxybenzoate

propyl p-hydroxybenzoate

butyl p-hydroxybenzoate

Example 4

A mouthwash formulation according to the present invention can be produced having the following composition: Water

Alcohol

Poloxamer 407

Sodium lauryl sulfate

SnF<2>/ACP and/or stabilized SnF<2>/ACFP

sodium fluoride

Flavors

sodium saccharin

Ethyl p-Hydroxybenzoate

Propyl P-Hydroxybenzoate

Butyl P-Hydroxybenzoate

Example 5

A sugar-free chewing gum formulation can be produced according to the present invention having the following composition:

Crystalline sorbitol/mannitol/xylitol

rubber base

Calcium carbonate

Glycerin

SnF<2>/ACP and/or stabilized SnF<2>/ACFP

sodium fluoride

Flavoring oil

Water

Example 6

The following is a protocol for the ionic analysis of a solution of ACP/SnF<2>stabilized with CPP. The total (firmly and weakly bound) and weakly bound samples were prepared as follows:

Total (firmly and weakly bound): One ml of 0.4% CPP-ACP/220 ppm F (as SnF2)/70 ppm F (as NaF) was taken and put into 19 ml of 1 M HNO3 and It was incubated at room temperature with slow and constant mixing from one end to the other for 24 hours (20 rpm). The mixture was centrifuged at 1,000 g for 15 minutes at room temperature. The supernatant was analyzed for calcium, tin, phosphate, and fluoride.

Weakly bound: A sample of 0.4% CPP-ACP/220 ppm F (as SnF2)/70 ppm F (as NaF) was taken and placed in a centricon with a 1,000 MWCO filter and centrifuged at 3,000 g for 1 hour at room temperature to produce sufficient filtrate (<10% of total sample so as not to affect equilibrium) for analysis by atomic absorption spectrophotometry (AAS) and ion chromatography (IC). The filtrates were then measured to give weakly bound ions.

Total and weakly bound calcium, tin, phosphate, and fluoride in the solution were determined by ion chromatography (for fluoride and phosphate) and atomic absorption spectrometry (for calcium and tin).

Ions strongly bound to CPP (colloidal retentate) were calculated from the difference between Total and weakly bound (as explained above). Based on this example, the tin-associated ACP complex had a stannous ion content of approximately 6 to 8 moles per mole of phosphopeptide.

Table 5. Ion analysis of 0.4% CPP-ACP/220 ppm F as SnF2/70 ppm F as NaF solution at pH 5.5 and pH 4.0 by atomic absorption spectrometry (for tin and calcium) and ion chromatography (for fluoride and phosphate).

Example 7

This in situ study was designed to compare remineralization using an established intraoral remineralization model promoted by five solutions:

1) amorphous calcium phosphate stabilized with calcium phosphopeptide (CPP-ACP) at 0.4% (w/v), 220 ppm stannous fluoride (SnF<2>) and 70 ppm sodium fluoride (NaF) (which shown in Figure 7 as CPP-ACP 2% SnF<2>); 2) CPP-ACP at 0.4% (w/v) and 290 ppm NaF;

3) CPP-ACP 0.4% (w/v);

4) 220 ppm SnF<2> and 70 ppm NaF without CPP-ACP (shown in Figure 7 as SnF<2>); and

5) 290 ppm NaF.

In this model, subjects wear a palatal appliance and rinse 4 times a day with 5 mL of solution for five consecutive 14-day treatment periods (including weekends) and rinse with a different solution during each treatment period. .

This randomized controlled study used a double-blind, five-way crossover design to evaluate the effects of five solutions to improve enamel remineralization using an intraoral remineralization model. Each solution contained the same amount of 290 ppm F (equivalent to 1,450 ppm F in a toothpaste diluted 1 in 5) [see above]. Subjects were randomly assigned to one of the five solutions for the first treatment period and, after a one-week rest period, transitioned to another solution for the second treatment period. This was repeated for the five treatments. Each subject wore a custom-made palatal appliance containing four pre-sterilized enamel plates containing artificially created subsurface lesions and rinsed four times a day for 14 consecutive days, including weekends (treatment period), for one minute with 5 mL of the assigned solution and then everything was expectorated. the solution and the accumulated saliva and continued using their device. The four rinses per day were performed (i) after breakfast, (ii) after lunch, (iii) after dinner, and (iv) before going to bed at night. After the first rinse per day of each treatment period, subjects on three different days expectorated the solution and collected saliva in a clean tube for ion analysis. The subjects kept a diary of the times and duration they rinsed with their solutions. Subjects were instructed to maintain their normal diet and oral hygiene procedures during the treatment periods. The appliances were removed during the subjects' normal oral hygiene procedures during the study period. After removing their braces for oral hygiene procedures, subjects cleaned their braces as instructed with a toothbrush and fluoride-free denture paste (both provided), avoiding areas of depressions, and gently rinsed their braces with DDW. before replacing them. Once out of the mouth, the appliances were stored in sealed, moist containers. All subjects brushed their teeth with standard fluoride toothpaste provided by the sponsor for the duration of the study. Subjects returned to the clinical site with their devices and diary at the end of each 14-day treatment period.

The researchers did not know what treatment had been administered or to the subject. Neither the investigators nor the registrar had access to the treatment code. Personnel dispensing the test solutions or supervising their use were not involved in the examination of the enamel half-blocks to minimize potential bias. After testing at baseline, subjects were assigned a subject number. Subject numbers were recorded on their case report forms. Subjects were randomly assigned to use one of the solutions during the first treatment period and then switched to another solution during the second to fifth treatment periods. Randomization was performed using a standard randomization table for the number of treatments in the crossover study. The results of the in situ study are shown in Figure 7. These results show that in an in situ model, a solution that includes complexes stabilized with tin-associated phosphopeptides (such as 2% CPP-ACP, with stannous fluoride and sodium fluoride, end column right, also referred to herein as SnF<2>/stabilized ACP) provide a significantly higher level of enamel subsurface remineralization compared to sodium fluoride alone, stannous fluoride and sodium fluoride, CPP-ACP alone or CPP-ACP with sodium fluoride. The two rightmost columns labeled 2% CPP-ACP+NaF and 2% CPP-ACP+SnF<2>were significantly different (p<0.01). This clearly demonstrates that in vivo in the oral cavity the complexes stabilized with tin-associated phosphopeptides of the invention have a superior capacity to remineralize lesions.

A palatal appliance was prepared for each subject by taking alginate impressions of the upper and lower dental arches from which study models were produced and articulated. For each subject, removable acrylic palatal appliances were made that covered the first premolars to the last tooth of the arch. The appliance consisted of a palatal plate that was retained in the mouth by four stainless steel hooks. The canals on each side of the appliance adjacent to the palatal surface of the maxillary premolar/molar teeth housed two half-blocks of enamel containing demineralized subsurface lesions. Therefore, each apparatus contained four half-blocks of enamel. The apparatus was designed to ensure that its surfaces were smooth and comfortable for the subjects.

Extracted third molars were obtained from the Royal Dental Hospital in Melbourne and from oral surgeons and general practitioners in private practice. All attached soft tissues were removed and the teeth were washed with distilled deionized water (DDW). All teeth in this study were sterilized by storing them in a 10% (v/v) neutral buffered formalin solution for at least two weeks at room temperature. After storage in formalin, the teeth were thoroughly washed in DDW and stored in DDW until needed. Healthy, relatively flat buccal and lingual surfaces of sterilized enamel with minimal cracking, staining, and fluorosis (as observed with a dissecting microscope) were selected and thoroughly rinsed again with DDW. The outer surface of the enamel, including surface cracks, was removed and then wet polished to a mirror finish using Soflex™ (3M) polishing discs on a low-speed contra-angle dental handpiece. Each polished surface of the tooth was then cut as a block of approximately 8 x 4 mm, using a water-cooled diamond disc saw, and the entire block was covered with acid-resistant nail varnish, except for two mesiodistal windows (occlusal and gingival) (approximately 1 x 7 mm) separated from each other by approximately 1 mm. Lesions were created in the enamel windows by mounting each block on the end of a 3 - 4 cm stick of yellow dental adhesive wax and immersing it in 40 mL of demineralization buffer without stirring, which consisted of 80 mL/L Noverite K -702 (polyacrylate, Lubrizol Corporation, Wickliffe, OH; White, 1987), 500 mg/L hydroxyapatite (Bio-Gel® HTP, Bio Rad Laboratories, Richmond CL), and 0.1 mol/L lactic acid (Ajax Chemicals, Auburn NSW) pH 4.8, for four days at 37°C. A solution change was performed after two days, at which time the blocks were removed from the solution, rinsed three times with DDW, dried, and placed in fresh demineralization buffer. The blocks were rinsed and dried in a similar manner after four days of demineralization. This demineralization procedure produces consistent subsurface lesions 80 to 110 μm deep with intact superficial layers, as assessed by microradiography of sections of the artificial lesions. After demineralization, each enamel block was cut across the midline of each window into two 4 × 4 mm blocks and the cut surface of each slab was covered with nail polish. Half of each block was retained as a demineralization control (half control block) and stored in a labeled 0.5 mL microcentrifuge tube along with a drop of DDW, thus creating a humidified environment. The other half of the enamel block was inserted into the intraoral appliance using dental wax for the remineralization protocol (half test block). Care was taken not to cover the artificial lesions with wax. At the end of an in situ treatment period, the enamel half-blocks were removed and replaced with new pre-sterilized enamel half-blocks for the beginning of a new test period. After each treatment period, each test half-block was paired with its control half-block and embedded, sectioned, and analyzed for mineral changes.

Each pair of polish half blocks (remineralization half block paired with its demineralization control half block) was carefully stripped of nail polish and rinsed thoroughly with distilled deionized water (DDW). The pair of polish half blocks ( test remineralization half block paired with its demineralization control half block) was placed on its cut sides in a small plastic vial with the lesion windows parallel and cold curing clear methacrylate resin (Paladur, Heraeus Kulzer) was poured , Germany) on the half blocks and allowed to set at room temperature. In order to identify the test and control half blocks, a permanent identification mark was drawn on the side of the methacrylate block next to the enamel half block. 200-300 μm thick sections were cut from the embedded block halves perpendicular to the lesion surface across the midline of both half-lesions using an internal ring saw microtome (Leitz 1600, Ernst Leitz). Wetzlar, Germany. The sections were then lapped at 80 ± 5 pm using a RotoPol-21/RotoForce4 lapping instrument (Struers, Denmark) with 1200 grit lapping paper. The lapped sections were removed from the lapping instrument and rinsed in DDW, They were dried and stored in a soft cloth between glass slides. Each section, containing the remineralized half-lesion and the paired demineralized control half-lesion from the same enamel block, was radiographed together with an aluminum wedge of 10 x 37.5 pm thick increments using high-resolution glass plates of Microchrome (3" x 3" x 0.06"; Microchrome, USA) and nickel-filtered copper Ka radiation at 20 kV and 30 mA for 8 minutes using an XMR microradiography system (Diffraction Technologies Pty Ltd) with a PANalytical fine-focusing glass , USA) for five minutes, placed in a glacial acetic acid stop bath for 30 seconds and fixed in Microchrome Developer F4 (1:4 dilution, Microchrome, USA) for five minutes. of all photochemicals was kept at 20 °C.

Radiographic images of the lesions were observed using transmitted light through a Dialux 20 microscope (Ernst Leitz Wetzlar, Germany). Images were acquired with a digital camera (Insight™) under the control of imaging software (Image Pro Plus version 7) running on a PC (Pentium III). Images of the lesions and neighboring areas of healthy enamel were scanned using the line luminance function of the program which provides readings in gray values between 0 and 256. An area free of artifacts or cracks was selected for analysis. Each scan comprised 200 readings taken from the surface of the tooth to the healthy enamel. The stepped wedge image was scanned on each slide and the averaged readings of the stepped gray value versus aluminum thickness were plotted. The tooth section image readings were within the linear portion of the step curve, and linear regression was used to convert the gray value data to aluminum equivalent thickness values. Section thicknesses were measured and % mineral data were calculated using the equation of Angmar et al. (1963) and the linear absorption coefficients of aluminum, organic matter plus water and the apatitic mineral (131.5, 11.3 and 260.5 respectively). The image of the median strip between the two lesions was scanned six times and averaged to obtain a control densitometric profile of healthy enamel. Lesion images (remineralization windows and demineralization control windows) on the gingival and occlusal side of the median strip were scanned in a similar manner, as close to the median strip as possible, but avoiding any irregularities commonly found in lesion edges, and % mineral profiles were calculated.

Example 8

In this example, several formulations are provided to exemplify the ways in which complexes of the invention can be formulated for compositions of different purposes as described more generally above. These are only examples of the type of formulations that can be provided using various embodiments of the invention.

Toothpaste formulations containing stabilized ACP or ACFP associated with tin Formulation 1

Formulation 2

Formulation 3

Formulation 4

Formulation 5

Formulation 6

Formulation 7

The invention disclosed and defined in this specification will be understood to extend to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

REFERENCES

• Aoba T, Fejerskov O (2002). Dentai fluorosis: chemistry and biology. Crit Rev Oral Biol Med 13:155-70. • Den Besten PK, Thariani H (1992). Biological mechanisms of fluorosis and level and timing of systemic exposure to fluoride with respect to fluorosis. J Dent Res 71:1238-43.

• Fejerskov O, Manji F, Baelum V (1990). The nature and mechanisms of dental fluorosis in man. J Dent Res 69 Spec No:692-700; discussion 721.

• Fejerskov O, Yanagisawa T, Tohda H, Larsen MJ, Josephsen K, Mosha HJ (1991). Posteruptive changes in human denial of fluorosis-a histological and ultrastructural study. Proc Finn Dent Soc 87:607-19.

• Giambro NJ, Prostak K, Den Besten PK (1995). Characterization of fluorosed human enamel by color reflectance, ultrastructure, and elemental composition. Caries Res 29:251-7.

• Reynolds EC (1998). Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: a review. Spec Care Dentist 18:8-16.

• Reynolds EC, Cai F, Shen P, Walker GD (2003). Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouthrinse or sugar-free chewing gum. J Dent Res 82:206-11.

Shen P, Cai F, Nowicki A, Vincent J, Reynolds EC (2001). Remineralization of enamel subsurface lesions by sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate. J Dent Res 80:2066-70.

Claims (18)

1. A complex of amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) stabilized with tin-associated phosphopeptide having a stannous ion content of at least 1 mole of tin per mole of phosphopeptide. 2. An amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) complex stabilized with tin-associated phosphopeptide according to claim 1, wherein the ACP and/or ACFP is stabilized with casein phosphopeptide. 3. An amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) complex stabilized with tin-associated phosphopeptide according to claim 1 or 2, wherein the complex has a stannous ion content of at least 5 moles, 6 moles, 7 moles, or about 8 moles of tin per mole of phosphopeptide. 4. A tin-associated phosphopeptide-stabilized amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) complex according to any one of claims 1 to 3, wherein the tin is provided by a selected compound. of the group consisting of stannous fluoride, stannous chloride, potassium stannous fluoride, sodium stannous fluorozirconate, stannous chloride fluoride, stannous acetate, sodium stannous fluoride, stannous hexafluorozirconate, stannous sulfate, stannous tartrate, stannous gluconate, disodium monotin citrate. 5. An amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) complex stabilized with tin-associated phosphopeptide according to claim 4, wherein the compound is stannous fluoride. 6. A method for producing a tin-associated phosphopeptide-stabilized amorphous calcium phosphate (ACP) or a tin-associated phosphopeptide-stabilized amorphous calcium fluoride phosphate (ACFP) comprising the step of mixing CPP-ACP or CPP-ACFP and a stannous compound in an aqueous solution, while maintaining the pH at about 6.5 or less. 7. An oral care formulation comprising an amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) complex stabilized with tin-associated phosphopeptide according to any one of claims 1 to 5. 8. An oral care formulation according to claim 7, wherein the composition is in the form applicable to the mouth selected from the group consisting of dentifrice including toothpaste, toothpaste powder and liquid dentifrice, mouthwash, mouth wash, oral spray, varnish, dental cement, chewing lozenges, chewing gum, toothpaste, gingival massage cream, gargle tablets, dairy products and other food products. 9. An oral care formulation according to claim 8, formulated as a toothpaste. 10. An oral care formulation according to claim 9, wherein the dentifrice is a toothpaste. 11. A tin-associated phosphopeptide-stabilized ACP and/or ACFP according to any one of claims 1 to 5 for use in the mineralization of a tooth surface or subsurface. 12. A complex of amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) stabilized with tin-associated phosphopeptide for use according to claim 11, wherein the tooth surface is an enamel lesion caused by cavities, dental erosion or fluorosis. 13. A complex of amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) stabilized with tin-associated phosphopeptide for use according to claim 11 or 12, wherein the tooth surface is exposed dentin, which which causes dentin hypersensitivity. 14. A complex of ACP or ACFP stabilized with tin-associated phosphopeptide for use in a method of forming a protective layer attached to the tooth surface, wherein the layer comprises calcium and phosphate, the method comprising contacting the surface or tooth subsurface with an ACP or ACFP stabilized with tin-associated phosphopeptide according to any one of claims 1 to 5. 15. A complex of ACP or ACFP stabilized with tin-associated phosphopeptide for use in a method according to claim 14, wherein the layer has a calcium:phosphate ratio equivalent to normal apatite. 16. A complex of ACP or ACFP stabilized with tin-associated phosphopeptide for use in a method according to claim 14 or 15, wherein the layer contains casein phosphopeptide peptides that stabilize the ACP or ACFP. 17. A complex of ACP or ACFP stabilized with tin-associated phosphopeptide for use in a method according to any one of claims 14 to 16, wherein the layer contains carbon, oxygen, fluoride, phosphate, calcium and tin. 18. A complex of ACP or ACFP stabilized with tin-associated phosphopeptide for use in a method according to claim 17, wherein the layer contains 20 to 30% by weight of carbon, 35 to 45% by weight of oxygen, 0.1 to 1% by weight of fluoride, 8 to 12% by weight of phosphate, 16 to 24% by weight of calcium and 0.5 to 2% by weight of tin.